Method of making crosslinked polymer matrices in tissue treatment applications

ABSTRACT

Crosslinked polymer compositions comprise a first synthetic polymer containing multiple nucleophilic groups covalently bound to a second synthetic polymer containing multiple electrophilic groups. The first synthetic polymer is preferably a synthetic polypeptide or a polyethylene glycol that has been modified to contain multiple nucleophilic groups, such as primary amino (—NH 2 ) or thiol (—SH) groups. The second synthetic polymer may be a hydrophilic or hydrophobic synthetic polymer which contains, or has been derivatized to contain, two or more electrophilic groups, such as succinimidyl groups. The compositions may further comprise other components, such as naturally occurring polysaccharides or proteins (such as glycosaminoglycans or collagen) and/or biologically active agents. Also disclosed are methods for using the crosslinked polymer compositions to effect adhesion between a first surface and a second surface; to effect tissue augmentation; to prevent the formation of surgical adhesions; and to coat a surface of a synthetic implant.

CROSS REFERENCES

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 08/573,799, filed Dec. 18, 1995, which application isincorporated herein by reference in full, and to which we claim priorityunder 35 U.S.C. § 120.

FIELD OF THE INVENTION

[0002] This invention relates generally to crosslinked polymercompositions comprising a first synthetic polymer containing multiplenucleophilic groups crosslinked using a second synthetic polymercontaining multiple electrophilic groups, and to methods of using suchcompositions as bioadhesives, for tissue augmentation, in the preventionof surgical adhesions, and for coating surfaces of synthetic implants,as drug delivery matrices and for ophthalmic applications.

BACKGROUND OF THE INVENTION

[0003] U.S. Pat. No. 5,162,430, issued Nov. 10, 1992, to Rhee et al.,and commonly owned by the assignee of the present invention, disclosescollagen-synthetic polymer conjugates prepared by covalently bindingcollagen to synthetic hydrophilic polymers such as various derivativesof polyethylene glycol.

[0004] Commonly owned U.S. Pat. No. 5,324,775, issued Jun. 28, 1994, toRhee et al., discloses various insert, naturally occurring,biocompatible polymers (such as polysaccharides) covalently bound tosynthetic, non-immunogenic, hydrophilic polyethylene glycol polymers.

[0005] Commonly owned U.S. Pat. No. 5,328,955, issued Jul. 12, 1994, toRhee et al., discloses various activated forms of polyethylene glycoland various linkages which can be used to produce collagen-syntheticpolymer conjugates having a range of physical and chemical properties.

[0006] Commonly owned, copending U.S. application Ser. No. 08/403,358,filed Mar. 14, 1995, discloses a crosslinked biomaterial compositionthat is prepared using a hydrophobic crosslinking agent, or a mixture ofhydrophilic and hydrophobic crosslinking agents. Preferred hydrophobiccrosslinking agents include any hydrophobic polymer that contains, orcan be chemically derivatized to contain, two or more succinimidylgroups.

[0007] Commonly owned, copending U.S. application Ser. No. 08/403,360,filed Mar. 14, 1995, discloses a composition useful in the prevention ofsurgical adhesions comprising a substrate material and an anti-adhesionbinding agent, where the substrate material preferably comprisescollagen and the binding agent preferably comprises at least onetissue-reactive functional group and at least one substrate-reactivefunctional group.

[0008] Commonly owned, U.S. application Ser. No. 08/476,825, filed Jun.7, 1995, by Rhee et al., discloses bioadhesive compositions comprisingcollagen crosslinked using a multifunctionally activated synthetichydrophilic polymer, as well as methods of using such compositions toeffect adhesion between a first surface and a second surface, wherein atleast one of the first and second surfaces is preferably a native tissuesurface.

[0009] Japanese patent publication No. 07090241 discloses a compositionused for temporary adhesion of a lens material to a support, to mountthe material on a machining device, comprising a mixture of polyethyleneglycol, having an average molecular weight in the range of 1000 - 5000,and poly-N-vinylpyrrolidone, having an average molecular weight in therange of 30,000 -200,000.

[0010] West and Hubbell, Biomaterials (1995) 16:1153-1156, disclose theprevention of post-operative adhesions using a photopolymerizedpolyethylene glycol-co-lactic acid diacrylate hydrogel and a physicallycrosslinked polyethylene glycol-co-polypropylene glycol hydrogel,Poloxamer 407®.

[0011] Each publication cited above and herein is incorporated herein byreference in its entirety to describe and disclose the subject matterfor which it is cited.

[0012] We now disclose a detailed description of preferred embodimentsof the present invention, including crosslinked polymer compositionscomprising synthetic polymers which contain multiple nucleophilic groupscrosslinked using synthetic polymers containing multiple electrophilicgroups, and methods for using these compositions to effect adhesionbetween a first surface and a second surface (wherein at least one ofthe first and second surfaces is preferably a native tissue surface) orto effect the augmentation of tissue, or to prevent surgical adhesion,or to coat surfaces of synthetic implants, or for delivering drugs orother active agents, or for ophthalmic applications.

SUMMARY OF THE INVENTION

[0013] The present invention discloses a crosslinked polymer compositioncomprising a first synthetic polymer containing two or more nucleophilicgroups, and a second synthetic polymer containing two or moreelectrophilic groups which are capable of covalently bonding to oneanother to form a three dimensional matrix.

[0014] A preferred composition of the invention comprises polyethyleneglycol containing two or more primary amino groups as the firstsynthetic polymer, and polyethylene glycol containing two or moresuccinimidyl groups (a five-membered ring structure represented hereinas —N(COCH₂)₂) as the second synthetic polymer.

[0015] In a general method for preparing a composition for the deliveryof a negatively charged compound (such as a protein or drug), a firstsynthetic polymer containing two or more nucleophilic groups is reactedwith a second synthetic polymer containing two or more electrophilicgroups, wherein the first synthetic polymer is present in molar excessin comparison to the second synthetic polymer, to form a positivelycharged matrix, which is then reacted with a negatively chargedcompound. In a general method for preparing a matrix for the delivery ofa positively charged compound, a first synthetic polymer containing twoor more nucleophilic groups is reacted with a second synthetic polymercontaining two or more electrophilic groups, wherein the secondsynthetic polymer is present in molar excess in comparison to the firstsynthetic polymer, to form a negatively charged matrix, which is thenreacted with a positively charged compound.

[0016] In a general method for effecting the nonsurgical attachment of afirst surface to a second surface, a first synthetic polymer containingtwo or more nucleophilic groups is mixed with a second synthetic polymercontaining two or more electrophilic groups to provide a reactionmixture; the reaction mixture is applied to a first surface beforesubstantial crosslinking has occurred; and the first surface iscontacted with a second surface to effect adhesion between the twosurfaces.

[0017] In a general method for augmenting soft or hard tissue within thebody of a mammalian subject, a first synthetic polymer containing two ormore nucleophilic groups and a second synthetic polymer containing twoor more electrophilic groups are administered simultaneously to a tissuesite in need of augmentation and the reaction mixture is allowed tocrosslink in situ to effect augmentation of the tissue. Alternatively,the first synthetic polymer and the second synthetic polymer may bemixed immediately prior to being administered to a tissue site, suchthat the majority of the crosslinking reaction proceeds in vivo.

[0018] In a general method for preventing the formation of adhesionsfollowing surgery, a first synthetic polymer containing two or morenucleophilic groups is mixed with a second synthetic polymer containingtwo or more electrophilic groups to provide a reaction mixture; thereaction mixture is applied to tissue comprising, surrounding, oradjacent to a surgical site before substantial crosslinking has occurredbetween the nucleophilic groups and the electrophilic groups; thereaction mixture is allowed to continue crosslinking in situ untilequilibrium crosslinking has been achieved; and the surgical site isclosed by conventional methodologies.

[0019] In a general method for coating a surface of a synthetic implant,a first synthetic polymer containing two or more nucleophilic groups ismixed with a second synthetic polymer containing two or moreelectrophilic groups to provide a reaction mixture; the reaction mixtureis applied to a surface of a synthetic implant; and the components ofthe reaction mixture are allowed to crosslink with each other on thesurface of the implant.

[0020] A feature of the invention is that the crosslinked polymercompositions are optically clear, making the compositions and methods ofthe invention particularly suited for use in ophthalmic applications inwhich optical clarity is a requirement. Furthermore, the compositions ofthe invention are comprised of biocompatible, non-immunogenic componentswhich leave no toxic, potentially inflammatory or immunogenic reactionproducts at the tissue site of administration.

[0021] Another feature of the invention is that the crosslinked polymercompositions have a high compression strength and high swellability,i.e., a composition that has been dried will swell to three times (ormore) its dried size upon rehydration, and is more “elastic.” Sincethese polymers are generally very hydrophilic, they are more easilyinjected, i.e., the crosslinked composition stays as a “cohesive mass”when injected through a fine gague (27-30 gague) needle.

[0022] Yet another feature of the invention is that nucleophilic groupson the first synthetic polymer may covalently bind to primary aminogroups on lysine residues of collagen molecules at the tissue site ofadministration, in effect, “biologically anchoring” the composition tothe host tissue.

[0023] One feature of the invention is that the components of thecompositions are non-immunogenic and do not require a “skin test” priorto beginning treatment, as do currently available xenogeneic collagencompositions, such as those manufactured from bovine hides.

[0024] Another feature of the invention is that, unlike collagen, thecompositions of the invention are not subject to enzymatic cleavage bymatrix metalloproteinases, such as collagenase, and are therefore notreadily degradable in vivo and, as such, are expected to have greaterlong-term persistence in vivo than prior art collagen compositions.

[0025] Still another feature is that, when the groups on each of thepolymers utilized react to form an amide bond, the manufacturing of thecompositions of the present invention can be highly controlled renderingmore consistent quality of products.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 shows compression force versus displacement for disks(approximate dimensions: 5 mm thick×5 mm diameter) of crosslinkedpolymer compositions comprising tetra-amino PEG (10,000 MW) crosslinkedusing tetrafunctionally activated SE-PEG

[0027] (10,000 MW), measured using the Instron Universal Tester, Model4202, at a compression rate of 2 mm per minute.

[0028]FIG. 2 shows compression force versus displacement for disks(approximate dimensions: 5 mm thick×5 mm diameter) of crosslinkedpolymer compositions comprising tetra-amino PEG (10,000 MW) crosslinkedusing trifunctionally activated SC-PEG (5,000 MW), measured using theInstron Universal Tester, Model 4202, at a compression rate of 2 mm perminute.

[0029]FIG. 3 shows the chemical structure of two commercially availablepolyethylene glycols containing multiple primary amino groups.

[0030] FIGS. 4 to 13 show the formation of various crosslinked syntheticpolymer compositions from hydrophilic polymers.

[0031] FIGS. 14 to 18 show the formation of various crosslinkedsynthetic polymer compositions from hydrophobic polymers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0032] In accordance with the present invention, crosslinked polymercompositions are prepared by reacting a first synthetic polymercontaining two or more nucleophilic groups with a second syntheticpolymer containing two or more electrophilic groups capable ofcovalently binding with the nucleophilic groups on the first syntheticpolymer.

[0033] The components of the present invention are non-immunogenic and,as such, do not require a “skin test” prior to starting treatment, asdoes xenogenic collagen. Also, unlike collagen, the compositions of theinvention are not subject to enzymatic cleavage by matrixmetalloproteinases (e.g., collagenase) and are therefore expected tohave greater long-term persistence in vivo than currently availablecollagen compositions.

[0034] The concept behind the present invention is that a syntheticpolymer containing multiple nucleophilic groups (represented below as“X”) will react with a synthetic polymer containing multipleelectrophilic groups (represented below as “Y”), resulting in acovalently bound polymer network, as follows:

polymer−X_(m)+polymer−Y_(n)→polymer−Z−polymer

[0035] wherein ≧2, n≧2,and m+n≧5;

[0036] X=—NH₂, —SH, —OH, —PH₂, —CO—NH—NH₂, etc., and can be the same ordifferent;

[0037] Y=—CO₂N(COCH₂)₂, —CO₂H, —CHO, —CHOCH₂, —N═C═O, SO₂CH═CH₂,—N(COCH)₂), —S—S—(C₅H₄N), etc., and can be the same or different; and

[0038] Z=functional group resulting from the union of a nucleophilicgroup (X) and an electrophilic group (Y).

[0039] As noted above, it is also contemplated by the present inventionthat X and Y may be the same or different, i.e., the polymer may havetwo different electrophilic groups, or two different nucleophilicgroups, such as with glutathione.

[0040] The backbone of each polymer is preferably an alkylene oxide,particularly, ethylene oxide, propylene oxide, and mixtures thereof.Examples of difunctional alkylene oxides can be represented by:X-polymer-X Y-polymer-Y

[0041] wherein X and Y are as defined above, and the term “polymer”represents - (CH₂CH₂O)_(n)- or -(CH(CH₃)CH₂O)_(n)- or-(CH₂CH₂O)_(n)-(CH(CH₃)CH₂O)_(n)

[0042] The required functional group X or Y is commonly coupled to thepolymer backbone by a linking group (represented below as “Q”), many ofwhich are known or possible. There are many ways to prepare the variousfunctionalized polymers, some of which are listed below: wherein Q =whole structure = —O—(CH₂)_(n)— polymer —O—(CH₂)_(n)—X (or Y)—S—(CH₂)_(n)— polymer —S—(CH₂)_(n)—X (or Y) —NH—(CH₂)_(n)— polymer—NH—(CH₂)_(n)—X (or Y) —O₂C—NH—(CH₂)_(n)— polymer —O₂C—NH—(CH₂)_(n)—X(or Y) —O₂C—(CH₂)_(n)— polymer —O₂C—(CH₂)_(n)—X (or Y) —O₂C—CR¹H—polymer —O₂C—CRH—X (or Y) —O—R²—CO—NH— polymer —O—R—CO—NH—X (or Y)

[0043] wherein n=1-10 in each case;

[0044] R¹=H, CH₃, C₂H₅, etc.;

[0045] R²=CH₂, CO—NH—CH₂ CH₂.

[0046] Q¹ and Q² may be the same or different.

[0047] For example, when Q² =OCH₂CH₂ (there is no Q¹ in this case);Y=—CO₂N(COCH₂)₂; and X=—NH₂, —SH, or —OH, the resulting reactions and Zgroups would be as follows:

polymer —NH₂ +polymer —OCH₂CH₂CO₂—N(COCH₂)₂→polymer—NH—OCH₂CH_(2(amide))

polymer —SH+polymer —OCH₂CH₂CO₂ —N(COCH₂)₂→polymer —S—OCH₂CH₂CO−polymer(thioester)

polymer —OH+polymer —OCH₂CH₂CO₂ —N(COCH₂)₂→polymer —O—OCH₂CH₂CO−p(ester)

[0048] An additional group, represented below as “D”, can be insertedbetween the polymer and the linking group to increase-degradation of thecrosslinked polymer composition in vivo, for example, for use in drugdelivery applications.

polymer —D—Q—X+polymer —D—Q—Y→polymer —D—Q—Z—Q—D -polymer-

[0049] Some useful biodegradable groups “D” include lactide, glycolide,ε-caprolactone, poly(α-hydroxy acid), poly(amino acids),poly(anhydride), and various di- or tripeptides.

[0050] Synthetic Polymers

[0051] In order to prepare the compositions of the present invention, itis first necessary to provide a first synthetic polymer containing twoor more nucleophilic groups, such as primary amino groups or thiolgroups, and a second synthetic polymer containing two or moreelectrophilic groups capable of covalently binding with the nucleophilicgroups on the second synthetic polymer, As used herein, the term“polymer” refers inter alia to polyalkyls, polyamino acids andpolysaccharides. Additionally, for external or oral use, the polymer maybe polyacrylic acid or carbopol.

[0052] As used herein, the term “synthetic polymer” refers to polymersthat are not naturally occurring and that are produced via chemicalsynthesis. As such, naturally occurring proteins such as collagen andnaturally occurring polysaccharides such as hyaluronic acid arespecifically excluded. Synthetic collagen, and synthetic hyaluronicacid, and their derivatives, are included. Synthetic polymers containingeither nucleophilic or electrophilic groups are also referred to hereinas “multifunctionally activated synthetic polymers”. The term“multifunctionally activated” (or, simply, “activated”) refers tosynthetic polymers which have, or have been chemically modified to have,two or more nucleophilic or electrophilic groups which are capable ofreacting with one another (i.e., the nucleophilic groups react with theelectrophilic groups) to form covalent bonds. Types of multifunctionallyactivated synthetic polymers include difunctionally activated,tetrafunctionally activated, and star-branched polymers.

[0053] Multifunctionally activated synthetic polymers for use in thepresent invention must contain at least two, more preferably, at leastthree, functional groups in order to form a three-dimensionalcrosslinked network with synthetic polymers containing multiplenucleophilic groups (i.e., “multi-nucleophilic polymers”). In otherwords, they must be at least difunctionally activated, and are morepreferably trifunctionally or tetrafunctionally activated. If the firstsynthetic polymer is a difunctionally activated synthetic polymer, thesecond synthetic polymer must contain three or more functional groups inorder to obtain a three- dimensional crosslinked network. Mostpreferably, both the first and the second synthetic polymer contain atleast three functional groups.

[0054] Synthetic Polymers Containing Multiple Nucleophilic Groups

[0055] Synthetic polymers containing multiple nucleophilic groups arealso referred to generically herein as “multi-nucleophilic polymers”.For use in the present invention, multi-nucleophilic polymers mustcontain at least two, more preferably, at least three, nucleophilicgroups. If a synthetic polymer containing only two nucleophilic groupsis used, a synthetic polymer containing three or more electrophilicgroups must be used in order to obtain a three-dimensional crosslinkednetwork.

[0056] Preferred multi-nucleophilic polymers for use in the compositionsand methods of the present invention include synthetic polymers thatcontain, or have been modified to contain, multiple nucleophilic groupssuch as primary amino groups and thiol groups. Preferredmulti-nucleophilic polymers include: (i) synthetic polypeptides thathave been synthesized to contain two or more primary amino groups orthiol groups; and (ii) polyethylene glycols that have been modified tocontain two or more primary amino groups or thiol groups. In general,reaction of a thiol group with an electrophilic group tends to proceedmore slowly than reaction of a primary amino group with an electrophilicgroup.

[0057] Preferred multi-nucleophilic polypeptides are syntheticpolypeptides that have been synthesized to incorporate amino acidscontaining primary amino groups (such as lysine) and/or amino acidscontaining thiol groups (such as cysteine). Poly(lysine), asynthetically produced polymer of the amino acid lysine (145 MW), isparticularly preferred. Poly(lysine)s have been prepared having anywherefrom 6 to about 4,000 primary amino groups, corresponding to molecularweights of about 870 to about 580,000.

[0058] Poly(lysine)s for use in the present invention preferably have amolecular weight within the range of about 1,000 to about 300,000; morepreferably, within the range of about 5,000 to about 100,000; mostpreferably, within the range of about 8,000 to about 15,000.Poly(lysine)s of varying molecular weights are commercially availablefrom Peninsula Laboratories, Inc. (Belmont, Calif.).

[0059] Polyethylene glycol can be chemically modified to containmultiple primary amino or thiol groups according to methods set forth,for example, in Chapter 22 of Poly(ethylene Glycol) Chemistry:Biotechnical and Biomedical Applications, J. Milton Harris, ed., PlenumPress, NY (1992). Polyethylene glycols which have been modified tocontain two or more primary amino groups are referred to herein as“multi-amino PEGs”. Polyethylene glycols which have been modified tocontain two or more thiol groups are referred to herein as “multi- thiolPEGs”. As used herein, the term “polyethylene glycol(s)” includesmodified and or derivatized polyethylene glycol(s).

[0060] Various forms of multi-amino PEG are commercially available fromShearwater Polymers (Huntsville, Ala.) and from Texaco Chemical Company(Houston, Tex.) under the name “Jeffamine”. Multi-amino PEGs useful inthe present invention include Texaco's Jeffamine diamines (“D” series)and triamines (“T” series), which contain two and three primary aminogroups per molecule, respectively. General structures for the Jeffiminediamines and triamines are shown in FIG. 3.

[0061] Polyamines such as ethylenediamine (H₂N—CH₂ CH₂—NH₂),tetramethylenediamine (H₂N-(CH₂)₄—NH₂), pentamethylenediamine(cadaverine) (H₂N-(CH₂)₅—NH₂), hexamethylenediamine (H₂N-(CH₂)₆—NH₂),bis(2-hydroxyethyl)amine (HN-(CH₂CH₂OH₂)₂), bis(2)aminoethyl)amine(HN-(CH₂CH₂NH₂)₂), and tris(2-aminoethyl)amine (N-(CH₂CH₂NH₂)₃) may alsobe used as the synthetic polymer containing multiple nucleophilicgroups.

[0062] Synthetic Polymers Containing Multiple Electrophilic Groups

[0063] Synthetic polymers containing multiple electrophilic groups arealso referred to herein as “multi-electrophilic polymers.” For use inthe present invention, the multifunctionally activated syntheticpolymers must contain at least two, more preferably, at least three,electrophilic groups in order to form a three-dimensional crosslinkednetwork with multi-nucleophilic polymers

[0064] Preferred multi-electrophilic polymers for use in thecompositions of the invention are polymers which contain two or moresuccinimidyl groups capable of forming covalent bonds with electrophilicgroups on other molecules. Succinimidyl groups are highly reactive withmaterials containing primary amino (—NH₂) groups, such as multi-aminoPEG, poly(lysine), or collagen. Succinimidyl groups are slightly lessreactive with materials containing thiol (—SH) groups, such asmulti-thiol PEG or synthetic polypeptides containing multiple cysteineresidues.

[0065] As used herein, the term “containing two or more succinimidylgroups” is meant to encompass polymers which are commercially availablecontaining two or more succinimidyl groups, as well as those that mustbe chemically derivatized to contain two or more succinimidyl groups. Asused herein, the term “succinimidyl group” is intended to encompasssulfosuccinimidyl groups and other such variations of the “generic”succinimidyl group. The presence of the sodium sulfite moiety on thesulfosuccinimidyl group serves to increase the solubility of thepolymer.

[0066] Hydrophilic Polymers

[0067] Hydrophilic polymers and, in particular, various polyethyleneglycols, are preferred for use in the compositions of the presentinvention. As used herein, the term “PEG” refers to polymers having therepeating structure (OCH₂ CH₂)_(n).

[0068] Structures for some specific, tetrafunctionally activated formsof PEG are shown in FIGS. 4 to 13, as are generalized reaction productsobtained by reacting tetrafunctionally activated PEGs with multi-aminoPEGs. As depicted in the Figures, the succinimidyl group is afive-member ring structure represented as —N(COCH₂)₂. In FIGS. 4 to 13,the symbol ΛΛΛ denotes an open linkage.

[0069]FIG. 4 shows the reaction of tetrafunctionally activated PEGsuccinimidyl glutarate, referred to herein as SG-PEG, with multi-aminoPEG, and the reaction product obtained thereby.

[0070] Another activated form of PEG is referred to as PEG succinimidylpropionate

[0071] (SE-PEG). The structural formula for tetrafunctionally activatedSE-PEG and the reaction product obtained by reacting it with multi-aminoPEG are shown in FIG. 5. In a general structural formula for thecompound, the subscript 3 is replaced with an “m”. In the embodimentshown in FIG. 4, m=3, in that there are three repeating CH₂ groups oneither side of the PEG.

[0072] The structure in FIG. 5 results in a conjugate which includes an“ether” linkage which is less subject to hydrolysis. This is distinctfrom the conjugate shown in FIG. 4, wherein an ester linkage isprovided. The ester linkage is subject to hydrolysis under physiologicalconditions.

[0073] Yet another functionally activated form of polyethylene glycol,wherein m=2, is shown in FIG. 6, as is the conjugate formed by reactingthe tetrafunctionally activated PEG with a multi-amino PEG.

[0074] Another functionally activated PEG similar to the compounds ofFIGS. 5 and 6 is provided when m=1. The structural formula of thetetrafunctionally activated PEG and resulting conjugate formed byreacting the activated PEG with multi-amino PEG are shown in FIG. 7. Itis noted that this conjugate includes both an ether and a peptidelinkage. These linkages are stable under physiological conditions.

[0075] Another functionally activated form of PEG is referred to as PEGsuccinimidyl succinamide (SSA-PEG). The structural formula for thetetrafunctionally activated form of this compound and the reactionproduct obtained by reacting it with multi-amino PEG are shown in FIG.8. In the structure shown in FIG. 8, m=2; however, related compounds,wherein m =1 or m =3 - 10, may also be used in the compositions of theinvention.

[0076] The structure in FIG. 8 results in a conjugate which includes an“amide” linkage which, like the ether linkage previously described, isless subject to hydrolysis and is therefore more stable than an esterlinkage.

[0077] Yet another activated form of PEG is provided when m=0. Thiscompound is referred to as PEG succinimidyl carbonate (SC-PEG). Thestructural formula of tetrafunctionally activated SC-PEG and theconjugate formed by reacting it with multi-amino PEG are shown in FIG.9.

[0078] As discussed above, preferred activated polyethylene glycolderivatives for use in the invention contain succinimidyl groups as thereactive group. However, different activating groups can be attached atsites along the length of the PEG molecule. For example, PEG can bederivatized to form functionally activated PEG propion aldehyde (A-PEG),the tetrafunctionally activated form of which is shown in FIG. 10, as isthe conjugate formed by the reaction of A-PEG with multi-amino PEG. Thelinkage shown in FIG. 10 is referred to as a -(CH₂)_(m)—NH—linkage,where m=1- 10.

[0079] Yet another form of activated polyethylene glycol is functionallyactivated PEG glycidyl ether (E-PEG), of which the tetrafunctionallyactivated compound is shown in FIG. 11, as is the conjugate formed byreacting such with multi-amino PEG.

[0080] Another activated derivative of polyethylene glycol isfunctionally activated PEG-isocyanate (I-PEG), which is shown in FIG. 2,along with the conjugate formed by reacting such with multi-amino PEG.

[0081] Another activated derivative of polyethylene glycol isfunctionally activated PEG-vinylsulfone (V-PEG), which is shown in FIG.13, below, along with the conjugate formed by reacting such withmulti-amino PEG.

[0082] Preferred multifunctionally activated polyethylene glycols foruse in the compositions of the present invention are polyethyleneglycols containing succinimidyl groups, such as SG-PEG and SE-PEG (shownin FIGS. 4 -7), preferably in trifunctionally or tetrafunctionallyactivated form.

[0083] Many of the activated forms of polyethylene glycol describedabove are now available commercially from Shearwater Polymers,Huntsville, Ala., and Union Carbide, South Charleston, W.Va.

[0084] Hydrophobic Polymers

[0085] Hydrophobic polymers can also be used to prepare the compositionsof the present invention. Hydrophobic polymers for use in the presentinvention preferably contain, or can be derivatized to contain, two ormore electrophilic groups, such as succinimidyl groups, most preferably,two, three, or four electrophilic groups. As used herein, the term“hydrophobic polymer” refers to polymers which contain a relativelysmall proportion of oxygen or nitrogen atoms.

[0086] Hydrophobic polymers which already contain two or moresuccinimidyl groups include, without limitation, disuccinimidyl suberate(DSS), bis(sulfosuccinimidyl) suberate (BS³),dithiobis(succinimidylpropionate) (DSP),bis(2-succinimidooxycarbonyloxy) ethyl sulfone (BSOCOES), and3,3′-dithiobis(sulfosuccinimidylpropionate (DTSPP), and their analogsand derivatives. The above-referenced polymers are commerciallyavailable from Pierce (Rockford, Ill.), under catalog Nos. 21555,21579,22585,21554, and 21577, respectively. Structural formulas for theabove-referenced polymers, as well as generalized reaction productsobtained by reacting each of these polymers with amino PEG, are shownbelow in FIGS. 14 -18, respectively.

[0087] Preferred hydrophobic polymers for use in the invention generallyhave a carbon chain that is no longer than about 14 carbons. Polymershaving carbon chains substantially longer than 14 carbons generally havevery-poor solubility in aqueous solutions and, as such, have very longreaction times when mixed with aqueous solutions of synthetic polymerscontaining multiple nucleophilic groups.

[0088] Derivatization of Polymers to Contain Functional Groups

[0089] Certain polymers, such as polyacids, can be derivatized tocontain two or more functional groups, such as succinimidyl groups.Polyacids for use in the present invention include, without limitation,trimethylolpropane-based tricarboxylic acid, di(trimethylolpropane)-based tetracarboxylic acid, heptanedioic acid, octanedioic acid(suberic acid), and hexadecanedioic acid (thapsic acid). Many of thesepolyacids are commercially available from DuPont Chemical Company.

[0090] According to a general method, polyacids can be chemicallyderivatized to contain two or more succinimidyl groups by reaction withan appropriate molar amount of

[0091] N-hydroxysuccinimide (NHS) in the presence ofN,N′-dicyclohexylcarbodiimide (DCC).

[0092] Polyalcohols such as trimethylolpropane and di(trimethylolpropane) can be converted to carboxylic acid form using various methods,then further derivatized by reaction with NHS in the presence of DCC toproduce trifunctionally and tetrafunctionally activated polymers,respectively, as described in commonly owned, copending U.S. applicationSer. No. 08/403,358. Polyacids such as heptanedioic acid(HOOC-(CH₂)₅—COOH), octanedioic acid (HOOC-(CH₂)₆—COOH), andhexadecanedioic acid (HOOC-(CH₂)₁₄—COOH) are derivatized by the additionof succinimidyl groups to produce difunctionally activated polymers.

[0093] Polyamines such as ethylenediamine (H₂N-CH₂ CH₂—NH₂),tetramethylenediamine (H₂N-(CH₂)₄—NH₂), pentamethylenediamine(cadaverine) (H₂N-(CH₂)₅—NH₂), hexamethylenediamine (H₂N-(CH₂)₆—NH₂),bis(2-hydroxyethyl)amine (HN-(CH₂CH₂OH)₂), bis(2)aminoethyl)amine(HN-(CH₂CH₂NH₂)₂), and tris(2-aminoethyl)amine (N-(CH₂CH₂NH₂)₃) can bechemically derivatized to polyacids, which can then be derivatized tocontain two or more succinimidyl groups by reacting with the appropriatemolar amounts of N-hydroxysuccinimide in the presence of DCC, asdescribed in U.S. application Ser. No. 08/403,358. Many of thesepolyamines are commercially available from DuPont Chemical Company.

[0094] Preparation of Crosslinked Polymer Compositions

[0095] In a general method for preparing the crosslinked polymercompositions of the invention, a first synthetic polymer containingmultiple nucleophilic groups is mixed with a second synthetic polymercontaining multiple electrophilic groups. Formation of athree-dimensional crosslinked network occurs as a result of the reactionbetween the nucleophilic groups on the first synthetic polymer and theelectrophilic groups on the second synthetic polymer.

[0096] Hereinafter, the term “first synthetic polymer” will be used torefer to a synthetic polymer containing two or more nucleophilic groups,and the term “second synthetic polymer” will be used to refer to asynthetic polymer containing two or more electrophilic groups. Theconcentrations of the first synthetic polymer and the second syntheticpolymer used to prepare the compositions of the present invention willvary depending upon a number of factors, including the types andmolecular weights of the particular synthetic polymers used and thedesired end use application.

[0097] In general, we have found that when using multi-amino PEG as thefirst synthetic polymer, it is preferably used at a concentration in therange of about 0.5 to about 20 percent by weight of the finalcomposition, while the second synthetic polymer is used at aconcentration in the range of about 0.5 to about 20 percent by weight ofthe final composition. For example, a final composition having a totalweight of 1 gram (1000 milligrams) would contain between about 5 toabout 200 milligrams of multi-amino PEG, and between about 5 to about200 milligrams of the second synthetic polymer.

[0098] Use of higher concentrations of both first and second syntheticpolymers will result in the formation of a more tightly crosslinkednetwork, producing a stiffer, more robust gel. As such, compositionsintended for use in tissue augmentation will generally employconcentrations of first and second synthetic polymer that fall towardthe higher end of the preferred concentration range. Compositionsintended for use as bioadhesives or in adhesion prevention do not needto be as firm and may therefore contain lower polymer concentrations.

[0099] Because polymers containing multiple electrophilic groups willalso react with water, the second synthetic polymer is generally storedand used in sterile, dry form to prevent the loss of crosslinkingability due to hydrolysis which typically occurs upon exposure of suchelectrophilic groups to aqueous media. Processes for preparing synthetichydrophilic polymers containing multiple electrophylic groups insterile, dry form are set forth in commonly assigned, copending U.S.application Ser. No. 08/497,573, filed Jun. 30, 1995. For example, thedry synthetic polymer may be compression molded into a thin sheet ormembrane, which can then be sterilized using gamma or, preferably,e-beam irradiation. The resulting dry membrane or sheet can be cut tothe desired size or chopped into smaller size particulates.

[0100] Polymers containing multiple nucleophilic groups are generallynot water- reactive and can therefore be stored in aqueous solution.

[0101] The crosslinked polymer compositions can also be prepared tocontain various imaging agents such as iodine or barium sulfate, orfluorine, in order to aid visualization of the compositions afteradministration via X-ray, or ¹⁹F-MRI, respectively.

[0102] Incorporation of Other Components into the Crosslinked SyntheticPolymer

[0103] Naturally occurring proteins, such as collagen, and derivativesof various naturally occurring polysaccharides, such asglycosaminoglycans, can additionally be incorporated into thecompositions of the invention. When these other components also containfunctional groups which will react with the functional groups on thesynthetic polymers, their presence during mixing and/or crosslinking ofthe first and second synthetic polymer will result in formation of acrosslinked synthetic polymer-naturally occurring polymer matrix. Inparticular, when the naturally occurring polymer (protein orpolysaccharide) also contains nucleophilic groups such as primary aminogroups, the electrophilic groups on the second synthetic polymer willreact with the primary amino groups on these components, as well as thenucleophilic groups on the first synthetic polymer, to cause these othercomponents to become part of the polymer matrix.

[0104] In general, glycosaminoglycans must be chemically derivatized bydeacetylation, desulfation, or both in order to contain primary aminogroups available for reaction with electrophilic groups on syntheticpolymer molecules. Glycosaminoglycans that can be derivatized accordingto either or both of the aforementioned methods include the following:hyaluronic acid, chondroitin sulfate A, chondroitin sulfate B (dermatansulfate), chondroitin sulfate C, chitin (can be derivatized tochitosan), keratan sulfate, keratosulfate, and heparin. Derivatizationof glycosaminoglycans by deacetylation and/or desulfation and covalentbinding of the resulting glycosaminoglycan derivatives with synthetichydrophilic polymers is described in further detail in commonlyassigned, allowed U.S. patent application Ser. No. 08/146,843, filedNov. 3, 1993.

[0105] Similarly, electrophilic groups on the second synthetic polymerwill react with primary amino groups on lysine residues or thiol groupson cysteine residues of certain naturally occurring proteins.Lysine-rich proteins such as collagen and its derivatives are especiallyreactive with electrophilic groups on synthetic polymers. As usedherein, the term “collagen” is intended to encompass collagen of anytype, from any source, including, but not limited to, collagen extractedfrom tissue or produced recombinantly, collagen analogues, collagenderivatives, modified collagens, and denatured collagens such asgelatin. Covalent binding of collagen to synthetic hydrophilic polymersis described in detail in commonly assigned U.S. Pat. No. 5,162,430,issued Nov. 10, 1992, to Rhee et al..

[0106] In general, collagen from any source may be used in thecompositions of the invention; for example, collagen may be extractedand purified from human or other mammalian source, such as bovine orporcine corium and human placenta, or may be recombinantly or otherwiseproduced. The preparation of purified, substantially non-antigeniccollagen in solution from bovine skin is well known in the art. Commonlyowned, U.S. Pat. No. 5,428,022, issued Jun. 27, 1995, to Palefsky etal., discloses methods of extracting and purifyng collagen from thehuman placenta. Commonly owned, copending U.S. application Ser. No.08/183,648, filed Jan. 18, 1994, discloses methods of producingrecombinant human collagen in the milk of transgenic animals, includingtransgenic cows. The term “collagen” or “collagen material” as usedherein refers to all forms of collagen, including those which have beenprocessed or otherwise modified.

[0107] Collagen of any type, including, but not limited to, types I, II,III, IV, or any combination thereof, may be used in the compositions ofthe invention, although type I is generally preferred. Eitheratelopeptide or telopeptide-containing collagen may be used; however,when collagen from a xenogeneic source, such as bovine collagen, isused, atelopeptide collagen is generally preferred, because of itsreduced immunogenicity compared to telopeptide-containing collagen.

[0108] Collagen that has not been previously crosslinked by methods suchas heat, irradiation, or chemical crosslinking agents is preferred foruse in the compositions of the invention, although previouslycrosslinked collagen may be used. Non-crosslinked atelopeptide fibrillarcollagen is commercially available from Collagen Corporation (Palo Alto,Calif.) at collagen concentrations of 35 mg/ml and 65 mg/ml under thetrademarks Zyderm® I Collagen and Zyderm II Collagen, respectively.Glutaraldehyde crosslinked atelopeptide fibrillar collagen iscommercially available from Collagen Corporation at a collagenconcentration of 35 mg/ml under the trademark Zyplast® Collagen.

[0109] Collagens for use in the present invention are generally inaqueous suspension at a concentration between about 20 mg/ml to about120 mg/ml; preferably, between about 30 mg/ml to about 90 mg/ml.

[0110] Although intact collagen is preferred, denatured collagen,commonly known as gelatin, can also be used in the compositions of theinvention. Gelatin may have the added benefit of being degradable fasterthan collagen.

[0111] Because of its tacky consistency, nonfibrillar collagen isgenerally preferred for use in compositions of the invention that areintended for use as bioadhesives. The term “nonfibrillar collagen”refers to any modified or unmodified collagen material that is insubstantially nonfibrillar form at pH 7, as indicated by optical clarityof an aqueous suspension of the collagen.

[0112] Collagen that is already in nonfibrillar form may be used in thecompositions of the invention. As used herein, the term “nonfibrillarcollagen” is intended to encompass collagen types that are nonfibrillarin native form, as well as collagens that have been chemically modifiedsuch that they are in nonfibrillar form at or around neutral pH.Collagen types that are nonfibrillar (or microfibrillar) in native forminclude types IV, VI, and VII.

[0113] Chemically modified collagens that are in nonfibrillar form atneutral pH include succinylated collagen and methylated collagen, bothof which can be prepared according to the methods described in U.S. Pat.No. 4,164,559, issued Aug. 14, 1979, to Miyata et al., which is herebyincorporated by reference in its entirety. Due to its inherenttackiness, methylated collagen is particularly preferred for use inbioadhesive compositions, as disclosed in commonly owned, U.S.application Ser. No. 08/476,825.

[0114] Collagens for use in the crosslinked polymer compositions of thepresent invention may start out in fibrillar form, then be renderednonfibrillar by the addition of one or more fiber disassembly agent. Thefiber disassembly agent must be present in an amount sufficient torender the collagen substantially nonfibrillar at pH 7, as describedabove. Fiber disassembly agents for use in the present inventioninclude, without limitation, various biocompatible alcohols, aminoacids, inorganic salts, and carbohydrates, with biocompatible alcoholsbeing-particularly preferred. Preferred biocompatible alcohols includeglycerol and propylene glycol. Non-biocompatible alcohols, such asethanol, methanol, and isopropanol, are not preferred for use in thepresent invention, due to their potentially deleterious effects on thebody of the patient receiving them. Preferred amino acids includearginine. Preferred inorganic salts include sodium chloride andpotassium chloride. Although carbohydrates, such as various sugarsincluding sucrose, may be used in the practice of the present invention,they are not as preferred as other types of fiber disassembly agentsbecause they can have cytotoxic effects in vivo.

[0115] Because it is opaque and less tacky than nonfibillar collagen,fibrillar collagen is less preferred for use in bioadhesivecompositions. However, as disclosed in commonly owned, U.S. applicationSer. No. 08/476,825, fibrillar collagen, or mixtures of nonfibrillar andfibrillar collagen, may be preferred for use in adhesive compositionsintended for long-term persistence in vivo, if optical clarity is not arequirement.

[0116] For compositions intended for use in tissue augmentation,fibrillar collagen is preferred because it tends to form strongercrosslinked gels having greater long-term persistency in vivo than thoseprepared using nonfibrillar collagen.

[0117] In general, the collagen is added to the first synthetic polymer,then the collagen and first synthetic polymer are mixed thoroughly toachieve a homogeneous composition. The second synthetic polymer is thenadded and mixed into the collagen / first synthetic polymer mixture,where it will covalently bind to primary amino groups or thiol groups onthe first synthetic polymer and primary amino groups on the collagen,resulting in the formation of a homogeneous crosslinked network. Variousdeacetylated and/or desulfated glycosaminoglycan derivatives can beincorporated into the composition in a similar manner as that describedabove for collagen.

[0118] For use in tissue adhesion as discussed below, it may also bedesirable to incorporate proteins such as albumin, fibrin or fibrinogeninto the crosslinked polymer composition to promote cellular adhesion.

[0119] In addition, the introduction of hydrocolloids such ascarboxymethylcellulose may promote tissue adhesion and/or swellability.

[0120] Administration of the Crosslinked Synthetic Polymer Compositions

[0121] The compositions of the present invention may be administeredbefore, during or after crosslinking of the first and second syntheticpolymer. Certain uses, which are discussed in greater detail below, suchas tissue augmentation, may require the compositions to be crosslinkedbefore administration, whereas other applications, such as tissueadhesion, require the compositions to be administered beforecrosslinking has reached “equilibrium.” The point at which crosslinkinghas reached equilibrium is defined herein as the point at which thecomposition no longer feels tacky or sticky to the touch.

[0122] In order to administer the composition prior to crosslinking, thefirst synthetic polymer and second synthetic polymer may be containedwithin separate barrels of a dual-compartment syringe. In this case, thetwo synthetic polymers do not actually mix until the point at which thetwo polymers are extruded from the tip of the syringe needle into thepatient's tissue. This allows the vast majority of the crosslinkingreaction to occur in situ, avoiding the problem of needle blockage whichcommonly occurs if the two synthetic polymers are mixed too early andcrosslinking between the two components is already too advanced prior todelivery from the syringe needle. The use of a dual-compartment syringe,as described above, allows for the use of smaller diameter needles,which is advantageous when performing soft tissue augmentation indelicate facial tissue, such as that surrounding the eyes.

[0123] Alternatively, the first synthetic polymer and second syntheticpolymer may be mixed according to the methods described above prior todelivery to the tissue site, then injected to the desired tissue siteimmediately (preferably, within about 60 seconds) following mixing.

[0124] In another embodiment of the invention, the first syntheticpolymer and second synthetic polymer are mixed, then extruded andallowed to crosslink into a sheet or other solid form. The crosslinkedsolid is then dehydrated to remove substantially all unbound water. Theresulting dried solid may be ground or comminuted into particulates,then suspended in a nonaqueous fluid carrier, including, withoutlimitation, hyaluronic acid, dextran sulfate, dextran, succinylatednoncrosslinked collagen, methylated noncrosslinked collagen, glycogen,glycerol, dextrose, maltose, triglycerides of fatty acids (such as cornoil, soybean oil, and sesame oil), and egg yolk phospholipid. Thesuspension of particulates can be injected through a small-gauge needleto a tissue site. Once inside the tissue, the crosslinked polymerparticulates will rehydrate and swell in size at least five-fold.

[0125] Use of Crosslinked Synthetic Polymers to Deliver ChargedCompounds

[0126] By varying the relative molar amounts of the first syntheticpolymer and the second synthetic polymer, it is possible to alter thenet charge of the resulting crosslinked polymer composition, in order toprepare a matrix for the delivery of a charged compound (such as aprotein or drug). As such, the delivery of charged proteins or drugs,which would normally diffuse rapidly out of a neutral carrier matrix,can be controlled.

[0127] For example, if a molar excess of a first synthetic polymercontaining multiple nucleophilic groups is used, the resulting matrixhas a net positive charge and can be used to ionically bind and delivernegatively charged compounds. Examples of negatively charged compoundsthat can be delivered from these matrices include various drugs, cells,proteins, and polysaccharides. Negatively charged collagens, such assuccinylated collagen, and glycosaminoglycan derivatives, such as sodiumhyaluronate, keratan sulfate, keratosulfate, sodium chondroitin sulfateA, sodium dermatan sulfate B, sodium chondroitin sulfate C, heparin,esterified chondroitin sulfate C, and esterified heparin, can beeffectively incorporated into the crosslinked polymer matrix asdescribed above.

[0128] If a molar excess of a second synthetic polymer containingmultiple electrophilic groups is used, the resulting matrix has a netnegative charge and can be used to ionically bind and deliver positivelycharged compounds. Examples of positively charged compounds that can bedelivered from these matrices include various drugs, cells, proteins,and polysaccharides. Positively charged collagens, such as methylatedcollagen, and glycosaminoglycan derivatives, such as esterifieddeacetylated hyaluronic acid, esterified deacetylated desulfatedchondroitin sulfate A, esterified deacetylated desulfated chondroitinsulfate C, deacetylated desulfated keratan sulfate, deacetylateddesulfated keratosulfate, esterified desulfated heparin, and chitosan,can be effectively incorporated into the crosslinked polymer matrix asdescribed above.

[0129] Use of Crosslinked Synthetic Polymers to Deliver BiologicallyActive Agents

[0130] The crosslinked polymer compositions of the present invention mayalso be used for localized delivery of various drugs and otherbiologically active agents. Biologically active agents such as growthfactors may be delivered from the composition to a local tissue site inorder to facilitate tissue healing and regeneration.

[0131] The term “biologically active agent” or “active agent” as usedherein refers to organic molecules which exert biological effects invivo. Examples of active agents include, without limitation, enzymes,receptor antagonists or agonists, hormones, growth factors, autogenousbone marrow, antibiotics, antimicrobial agents and antibodies. The term“active agent” is also intended to encompass various cell types andgenes which can be incorporated into the compositions of the invention.The term “active agent” is also intended to encompass combinations ormixtures of two or more active agents, as defined above.

[0132] Preferred active agents for use in the compositions of thepresent invention include growth factors, such as transforming growthfactors (TGFs), fibroblast growth factors (FGFs), platelet derivedgrowth factors (PDGFs), epidermal growth factors (EGFs), connectivetissue activated peptides (CTAPs), osteogenic factors, and biologicallyactive analogs, fragments, and derivatives of such growth factors.Members of the transforming growth factor (TGF) supergene family, whichare multifunctional regulatory proteins, are particularly preferred.Members of the TGF supergene family include the beta transforming growthfactors (for example, TGF-β1, TGF-β2, TGF-β3); bone morphogeneticproteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7,BMP-8, BMP-9); heparin-binding growth factors (for example, fibroblastgrowth factor (FGF), epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF)); Inhibins (forexample, Inhibin A, Inhibin B); growth differentiating factors (forexample, GDF-1); and Activins (for example, Activin A, Activin B,Activin AB).

[0133] Growth factors can be isolated from native or natural sources,such as from mammalian cells, or can be prepared synthetically, such asby recombinant DNA techniques or by various chemical processes. Inaddition, analogs, fragments, or derivatives of these factors can beused, provided that they exhibit at least some of the biologicalactivity of the native molecule. For example, analogs can be prepared byexpression of genes altered by site-specific mutagenesis or othergenetic engineering techniques.

[0134] Biologically active agents may be incorporated into thecrosslinked synthetic polymer composition by admixture. Alternatively,the agents may be incorporated into the crosslinked polymer matrix, asdescribed above, by binding these agents with the functional groups onthe synthetic polymers. Processes for covalently binding biologicallyactive agents such as growth factors using functionally activatedpolyethylene glycols are described in commonly assigned U.S. Pat. No.5,162,430, issued Nov. 10, 1992, to Rhee et al.. Such compositionspreferably include linkages that can be easily biodegraded, for exampleas a result of enzymatic degradation, resulting in the release of theactive agent into the target tissue, where it will exert its desiredtherapeutic effect.

[0135] A simple method for incorporating biologically active agentscontaining nucleophilic groups into the crosslinked polymer compositioninvolves mixing the active agent with the first synthetic polymer (orfirst synthetic polymer / collagen mixture) prior to adding the secondsynthetic polymer. This procedure will result in covalent binding of theactive agent to the crosslinked polymer composition, producing a highlyeffective sustained release composition.

[0136] The type and amount of active agent used will depend, among otherfactors, on the particular site and condition to be treated and thebiological activity and pharmacokinetics of the active agent selected.

[0137] Use of Crosslinked Synthetic Polymers to Deliver Cells or Genes

[0138] The crosslinked polymer compositions of the present invention canalso be used to deliver various types of living cells or genes to adesired site of administration in order to form new tissue. The term“genes” as used herein is intended to encompass genetic material fromnatural sources, synthetic nucleic acids, DNA, antisense-DNA and RNA.

[0139] When used to deliver cells, for example, mesenchymal stem cellscan be delivered to produce cells of the same type as the tissue intowhich they are delivered. Mesenchymal stem cells are not differentiatedand therefore can differentiate to form various types of new cells dueto the presence of an active agent or the effects (chemical, physical,etc.) of the local tissue environment. Examples of mesenchymal stemcells include osteoblasts, chondrocytes, and fibroblasts. Osteoblastscan be delivered to the site of a bone defect to produce new bone;chondrocytes can be delivered to the site of a cartilage defect toproduce new cartilage; fibroblasts can be delivered to produce collagenwherever new connective tissue is needed; neurectodermal cells can bedelivered to form new nerve tissue; epithelial cells can be delivered toform new epithelial tissues, such as liver, pancreas, etc..

[0140] The cells or genes may be either allogeneic or xenogeneic inorigin. For example, the compositions can be used to deliver cells orgenes from other species which have been genetically modified. Becausethe compositions of the invention are not easily degraded in vivo, cellsand genes entrapped within the crosslinked polymer compositions will beisolated from the patient's own cells and, as such, will not provoke animmune response in the patient. In order to entrap the cells or geneswithin a crosslinked polymer matrix, the first polymer and the cells orgenes may be pre-mixed, then the second polymer is mixed into the firstpolymer/cell or gene mixture to form a crosslinked matrix, therebyentrapping the cells or genes within the matrix.

[0141] As discussed above for biologically active agents, when used todeliver cells or genes, the synthetic polymers preferably also containbiodegradable groups to aid in controlled release of the cells or genesat the intended site of delivery.

[0142] Use of the Crosslinked Synthetic Polymers as Bioadhesives

[0143] We have found that the preferred compositions of the inventiontend to have unusually high tackiness, making them particularly suitablefor use as bioadhesives, for example, for use in surgery. As usedherein, the terms “bioadhesive”, “biological adhesive”, and “surgicaladhesive” are used interchangeably to refer to biocompatiblecompositions capable of effecting temporary or permanent attachmentbetween the surfaces of two native tissues, or between a native tissuesurface and a non-native tissue surface or a surface of a syntheticimplant.

[0144] In a general method for effecting the attachment of a firstsurface to a second surface, the first synthetic polymer and the secondsynthetic polymer are applied to a first surface, then the first surfaceis contacted with a second surface to effect adhesion between the firstsurface and the second surface. Preferably, the first synthetic polymerand second synthetic polymer are first mixed to initiate crosslinking,then delivered to a first surface before substantial crosslinking hasoccurred between the nucleophilic groups on the first synthetic polymerand the electrophilic groups on the second synthetic polymer. The firstsurface is then contacted with the second surface, preferablyimmediately, to effect adhesion between the two surfaces. At least oneof the first and second surfaces is preferably a native tissue surface.

[0145] For example, the first synthetic polymer and second syntheticpolymer are generally provided in separate syringes, the contents ofwhich-are then mixed together using syringe-to-syringe mixing techniquesjust prior to delivery to a first surface. The first synthetic polymerand second synthetic polymer are preferably mixed for a minimum of 20(preferably, 20 to 100; more preferably, 30 to 60) passes to ensureadequate mixing. As crosslinking between the corresponding reactivegroups on the two synthetic polymers is generally initiated during themixing process, it is important to deliver the reaction mixture to thefirst surface as soon as possible after mixing.

[0146] The reaction mixture can be extruded onto the first surface fromthe opening of a syringe or other appropriate extrusion device.Following application, the extruded reaction mixture can be spread overthe first surface using a spatula, if necessary. Alternatively, thefirst synthetic polymer and the second synthetic polymer can be mixedtogether in an appropriate mixing dish or vessel, then applied to thefirst surface using a spatula.

[0147] In another method for preparing the reaction mixture, the firstsynthetic polymer and second synthetic polymer are contained in separatechambers of a spray can or bottle with a nozzle, or other appropriatespraying device. In this scenario, the first and second polymers do notactually mix until they are expelled together from the nozzle of thespraying device. Following application of the reaction mixture to asurface containing collagen, the first surface is contacted with asecond surface. If the two surfaces are contacted before substantialcrosslinking has occurred between the synthetic polymer and thecrosslinking agent, the reactive groups on the crosslinking agent willalso covalently bond with primary amino groups on lysine residues ofcollagen molecules present on either or both of the surfaces, providingimproved adhesion.

[0148] The two surfaces may be held together manually, or using otherappropriate means, while the crosslinking reaction is proceeding tocompletion. Crosslinking is typically complete within 5 to 60 minutesafter mixing of the first and second synthetic polymers. However, thetime required for complete crosslinking to occur is dependent on anumber of factors, including the types and molecular weights of the twosynthetic polymers and, most particularly, the concentrations of the twosynthetic polymers (i.e., higher concentrations result in fastercrosslinking times).

[0149] At least one of the first and second surfaces is preferably anative tissue surface. As used herein, the term “native tissue” refersto biological tissues that are native to the body of the specificpatient being treated. As used herein, the term “native tissue” isintended to include biological tissues that have been elevated orremoved from one part of the body of a patient for implantation toanother part of the body of the same patient (such as bone autografts,skin flap autografts, etc.). For example, the compositions of theinvention can be used to adhere a piece of skin from one part of apatient's body to another part of the body, as in the case of a burnvictim.

[0150] The other surface may be a native tissue surface, a non-nativetissue surface, or a surface of a synthetic implant. As used herein, theterm “non-native tissue” refers to biological tissues that have beenremoved from the body of a donor patient (who may be of the same speciesor of a different species than the recipient patient) for implantationinto the body of a recipient patient (e.g., tissue and organtransplants). For example, the crosslinked polymer compositions of thepresent invention can be used to adhere a donor cornea to the eye of arecipient patient.

[0151] As used herein, the term “synthetic implant” refers to anybiocompatible material intended for implantation into the body of apatient not encompassed by the above definitions for native tissue andnon-native tissue. Synthetic implants include, for example, artificialblood vessels, heart valves, artificial organs, bone prostheses,implantable lenticules, vascular grafts, stents, and stent/graftcombinations, etc..

[0152] Use of Crosslinked Synthetic Polymers in Ophthalmic Applications

[0153] Because of their optical clarity, the crosslinked polymercompositions of the invention which do not contain collagen areparticularly well suited for use in ophthalmic applications. Forexample, a synthetic lenticule for correction of vision can be attachedto the Bowman's layer of the cornea of a patient's eye using the methodsof the present invention. As disclosed in commonly assigned, allowedU.S. application Ser. No. 08/147,227, filed Nov. 3,1993, by Rhee et al.,a chemically modified collagen (such as succinylated or methylatedcollagen) which is in substantially nonfibrillar form at pH 7 can becrosslinked using a synthetic hydrophilic polymer, then molded into adesired lenticular shape and allowed to complete crosslinking. Theresulting crosslinked collagen lenticule can then be attached to theBowman's layer of a de-epithelialized cornea of a patient's eye usingthe methods of the present invention. By applying the reaction mixturecomprising the first and second synthetic polymers to the anteriorsurface of the cornea, then contacting the anterior surface of thecornea with the posterior surface of the lenticule before substantialcrosslinking has occurred, electrophilic groups on the second syntheticpolymer will also covalently bind with collagen molecules in both thecorneal tissue and the lenticule to firmly anchor the lenticule inplace. (Alternatively, the reaction mixture can be applied first to theposterior surface of the lenticule, which is then contacted with theanterior surface of the cornea.)

[0154] The compositions of the present invention are also suitable foruse in vitreous replacement.

[0155] Use of Crosslinked Synthetic Polymer Compositions in TissueAugmentation

[0156] The crosslinked polymer compositions of the invention can also beused for augmentation of soft or hard tissue within the body of amammalian subject.

[0157] As such, they may be better than currently marketedcollagen-based materials product for soft tissue augmentation, becausethey are less immunogenic and more persistent. Examples of soft tissueaugmentation applications include sphincter (e.g., urinary, anal,esophageal) sphincter augmentation and the treatment of rhytids andscars. Examples of hard tissue augmentation applications include therepair and/or replacement of bone and/or cartilaginous tissue.

[0158] The compositions of the invention are particularly suited for useas a replacement material for synovial fluid in osteoarthritic joints,where the crosslinked polymer compositions serve to reduce joint painand improve joint function by restoring a soft hydrogel network in thejoint. The crosslinked polymer compositions can also be used as areplacement material for the nucleus pulposus of a damagedintervertebral disk. As such, the nucleus pulposus of the damaged diskis first removed, then the crosslinked polymer composition is injectedor otherwise introduced into the center of the disk. The composition mayeither be crosslinked prior to introduction into the disk, or allowed tocrosslink in situ.

[0159] In a general method for effecting augmentation of tissue withinthe body of a mammalian subject, the first and second synthetic polymersare injected simultaneously to a tissue site in need of augmentationthrough a small-gauge (e.g., 25 - 32 gauge) needle. Once inside thepatient's body, the nucleophilic groups on the first synthetic polymerand the electrophilic groups on the second synthetic polymer will reactwith each other to form a crosslinked polymer network in situ.Electrophilic groups on the second synthetic polymer may also react withprimary amino groups on lysine residues of collagen molecules within thepatient's own tissue, providing for “biological anchoring” of thecompositions with the host tissue.

[0160] Use of the Crosslinked Synthetic Polymer Compositions to PreventAdhesions

[0161] Another use of the crosslinked polymer compositions of theinvention is to coat tissues in order to prevent the formation ofadhesions following surgery or injury to internal tissues or organs. Ina general method for coating tissues to prevent the formation ofadhesions following surgery, the first and second synthetic polymers aremixed, then a thin layer of the reaction mixture is applied to thetissues comprising, surrounding, and/or adjacent to the surgical sitebefore substantial crosslinking has occurred between the nucleophilicgroups on the first synthetic polymer and the electrophilic groups onthe second synthetic polymer. Application of the reaction mixture to thetissue site may be by extrusion, brushing, spraying (as describedabove), or by any other convenient means.

[0162] Following application of the reaction mixture to the surgicalsite, crosslinking is allowed to continue in situ prior to closure ofthe surgical incision. Once crosslinking has reached equilibrium,tissues which are brought into contact with the coated tissues will notstick to the coated tissues. At this point in time, the surgical sitecan be closed using conventional means (sutures, etc.).

[0163] In general, compositions that achieve complete crosslinkingwithin a relatively short period of time (i.e., 5 - 15 minutes followingmixture of the first synthetic polymer and the second synthetic polymer)are preferred for use in the prevention of surgical adhesions, so thatthe surgical site may be closed relatively soon after completion of thesurgical procedure.

[0164] Use of the Crosslinked Synthetic Polymers to Coat Implants

[0165] Another use of the crosslinked polymer compositions of theinvention is as a coating material for synthetic implants. In a generalmethod for coating a surface of a synthetic implant, the first andsecond synthetic polymers are mixed, then a thin layer of the reactionmixture is applied to a surface of the implant before substantialcrosslinking has occurred between the nucleophilic groups on the firstsynthetic polymer and the electrophilic groups on the second syntheticpolymer. In order to minimize cellular and fibrous reaction to thecoated implant, the reaction mixture is preferably prepared to have anet neutral charge. Application of the reaction mixture to the implantsurface may be by extrusion, brushing, spraying (as described above), orby any other convenient means. Following application of the reactionmixture to the implant surface, crosslinking is allowed to continueuntil complete crosslinking has been achieved.

[0166] Although this method can be used to coat the surface of any typeof synthetic implant, it is particularly useful for implants wherereduced thrombogenicity is an important consideration, such asartificial blood vessels and heart valves, vascular grafts, vascularstents, and stent/graft combinations. The method may also be used tocoat implantable surgical membranes (e.g., monofilament polypropylene)or meshes (e.g., for use in hernia repair). Breast implants may also becoated using the above method in order to minimize capsular contracture.

[0167] The compositions of the present invention may also be used tocoat lenticules, which are made from either naturally occurring orsynthetic polymers.

[0168] Use of the Crosslinked Synthetic Polymers to Treat Aneurism

[0169] The crosslinked polymer compositions of the invention can beextruded or molded in the shape of a string or coil, then dehydrated.The resulting dehydrated string or coil can be delivered via catheter tothe site of a vascular malformation, such as an aneurysm, for thepurpose of vascular occlusion and, ultimately, repair of themalformation. The dehydrated string or coil can be delivered in acompact size and will rehydrate inside the blood vessel, swellingseveral times in size compared to its dehydrated state, whilemaintaining its original shape.

[0170] Other Uses for the Crosslinked Synthetic Polymers

[0171] As discussed in commonly assigned, copending application Ser. No.08/574,050, filed Dec. 18, 1995, which is incorporated herein byreference, the crosslinked polymer compositions of the invention can beused to block or fill various lumens and voids in the body of amammalian subject. The compositions can also be used as biosealants toseal fissures or crevices within a tissue or structure (such as avessel), or junctures between adjacent tissues or structures, to preventleakage of blood or other biological fluids.

[0172] The crosslinked polymer compositions can also be used as a largespace-filling device for organ displacement in a body cavity duringsurgical or radiation procedures, for example, to protect the intestinesduring a planned course of radiation to the pelvis.

[0173] The crosslinked polymer compositions of the invention can also becoated onto the interior surface of a physiological lumen, such as ablood vessel or Fallopian tube, thereby serving as a sealant to preventrestenosis of the lumen following medical treatment, such as, forexample, balloon catheterization to remove arterial plaque deposits fromthe interior surface of a blood vessel, or removal of scar tissue orendometrial tissue from the interior of a Fallopian tube. A thin layerof the reaction mixture is preferably applied to the interior surface ofthe vessel (for example, via catheter) immediately following mixing ofthe first and second synthetic polymers. Because the compositions of theinvention are not readily degradable in vivo, the potential forrestenosis due to degradation of the coating is minimized. The use ofcrosslinked polymer compositions having a net neutral charge furtherminimizes the potential for restenosis.

EXAMPLES

[0174] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make the preferred embodiments of the conjugates, compositions,and devices and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature,molecular weight, etc.) but some experimental errors and deviationshould be accounted for. Unless indicated otherwise, parts are parts byweight, molecular weight is weight average molecular weight, temperatureis in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1 (Preparation of Crosslinked Multi-amino PEG Compositions)

[0175] 0.15 grams of di-amino PEG (3400 MW, obtained from ShearwaterPolymers, Huntsville, Ala) in 250 ul of water was mixed with 0.1 g oftrifunctionally activated SC-PEG (5000 MW, also obtained from ShearwaterPolymers) using syringe-to-syringe mixing. The reaction mixture wasextruded onto a petri dish and formed a soft gel at room temperature.

[0176] 0.15 gram of di-amino PEG in 250 μl of water was mixed with 0.1 gof tetrafunctionally activated SE-PEG (also from Shearwater Polymers)using syringe-to-syringe mixing. The reaction mixture was extruded ontoa petri dish and formed a soft gel at room temperature.

EXAMPLE 2 (Preparation of Crosslinked Multi-amino PEG Compositions)

[0177] The following stock solutions of various di-amino PEGs wereprepared:

[0178] Ten (10) grams of Jeffamine ED-2001 (obtained from TexacoChemical Company, Houston, Tex.) was dissolved in 9 ml of water.

[0179] Ten (10) grams of Jeffamine ED-4000 (also obtained from TexacoChemical Company) was dissolved in 9 ml of water. 0.1 grams of di-aminoPEG (3400 MW, obtained from Shearwater Polymers, Huntsville, Ala.) wasdissolved in 300 μl of water.

[0180] Each of the three di-amino PEG solutions prepared above was mixedwith aqueous solutions of trifunctionally activated SC-PEG (TSC-PEQ,5000 MW, also obtained from Shearwater Polymers) as set forth in Table1, below. TABLE 1 Preparation of Crosslinked Polymer CompositionsDi-amino PEG TSC-PEG + Aqueous Solvent 50 ul   0 mg + 50 μl water 50 ul10 mg + 50 μl PBS  50 ul 10 mg + 100 μl PBS 250 ul  50 mg + 500 μl PBS

[0181] The solutions of di-amino PEG and TSC-PEG were mixed usingsyringe-to-syringe mixing. Each of the materials was extruded from thesyringe and allowed to set for 1 hour at 37° C. Each of the materialsformed a gel. In general, the gels became softer with increasing watercontent; the gels containing the least amount of aqueous solvent (wateror PBS) were firmest.

EXAMPLE 3 (Characterization of Crosslinked Multi-amino PEG Compositions)

[0182] Fifty (50) milligrams of tetra-amino PEG (10,000 MW, obtainedfrom Shearwater Polymers, Huntsville, Ala.) in 0.5 ml PBS was mixed,using syringe-to-syringe mixing, with 50 mg of tetrafunctionallyactivated SE-PEG (“tetra SE-PEG”, 10,000 MW, also obtained fromShearwater Polymers) in 0.5 ml PBS or trifunctionally activated SC-PEG(“tri SC-PEG”, 5000 MW, also obtained from Shearwater Polymers) in 0.5ml PBS.

[0183] Syringes containing each of the two mixtures were incubated at37° C. for approximately 16 hours. Both compositions formed elasticgels. The gels were pushed out of the syringes and sliced into 5 - 6 mmthick disks having a diameter of 5 mm, for use in compression andswellability testing, as described below.

[0184] Compression force versus displacement for the two gels wasmeasured in the Instron Universal Tester, Model 4202, at a compressionrate of 2 mm per minute, using disks of the two gels prepared asdescribed above. Compression force (in Newtons) versus gel displacement(in millimeters) is shown in FIGS. 1 and 2 for gels prepared using thetetra SE-PEG and tri SC-PEG, respectively.

[0185] Under compression forces as high as 30 - 35 Newtons, the gels didnot break, but remained elastic.

[0186] Disks of each of the two gels, prepared as described above, wereweighed and the dimensions (diameter and length) measured. The diskswere then immersed in PBS and incubated at 37° C. After 3 daysincubation, the disks were removed from the PBS, weighed, and measured.Results of swellability testing are shown in Table 2, below. TABLE 2Swellability Testing of Crosslinked Multi-amino PEG CompositionsDimensions (in mm) Gel Weight (in grams) (diameter/thickness)Crosslinking Before After Before After Agent Swelling Swelling SwellingSwelling Tetra SE-PEG 0.116 0.310 5.0/5.0 7.1/8.1 Tri SC-PEG 0.131 0.2875.0/6.0 6.4/8.5

[0187] As shown above, the gels swelled two to three times in weight, aswell as swelling an average of about 50% in both diameter and thickness.

EXAMPLE 4 (Preparation of Crosslinked Poly(lysine) Compositions)

[0188] Ten (10) milligrams of poly-L-lysine hydrobromide (8,000 MW,obtained from Peninsula Laboratories, Belmont, Calif.) in 0.1 mlphosphate buffer (0.2 M, pH=6.6) was mixed with 10 mg oftetraflnctionally activated SE-PEG (10,000 MW, obtained from ShearwaterPolymers, Huntsville, Ala.) in 0.1 ml PBS. The composition formed a softgel almost immediately.

EXAMPLE 5 (Preparation and Mechanical Testing of CrosslinkedMulti-amnino PEG Compositions)

[0189] Gels comprising tetra-amino PEG (10,000 MW, obtained fromShearwater Polymers, Huntsville, Ala.) and 14% (by weight) oftetrafunctionally activated SE-PEG (“tetra SE-PEG”, 10,000 MW, alsoobtained from Shearwater Polymers) were prepared by mixing thetetra-amino PEG (at a concentration of 25 mg/ml in water) with the tetraSE-PEG (in PBS) in a petri dish. The resulting tetra-amino PEG / SE-PEGmixtures were incubated for 16 hours at 37° C.

[0190] The mixture containing 1% SE-PEG did not form a gel due to thelow SE-PEG concentration. The mixture containing 2% SE-PEG formed a gelat some point during the 16-hour incubation period. The mixturescontaining 3 and 4% SE-PEG formed gels within approximately 46 minutesof mixing. The gel containing 2% SE-PEG was readily extrudable through a30-gauge needle; the gel containing 3% SE-PEG could be extruded througha 27-gauge needle.

[0191] The effect of elevated temperature on gel formation wasevaluated. Gels comprising tetra-amino PEG and 2.5% (by weight) tetraSE-PEG were prepared and incubated at temperatures of 37° C and 40-50°C. Elevated temperature was found to have a marked effect on gelationtime: the tetra-amino PEG / SE-PEG mixture incubated at 37° C. formed agel within approximately 20-25 minutes, whereas mixtures incubated at40-50° C formed gels within approximately 5 minutes. Both gels wereextrudable through a 27-gauge needle. The effect of pH on gel formationwas evaluated. Gels comprising tetra-amino PEG and 2.5% (by weight)tetra SE-PEG were prepared as set forth in Table 3, below. TABLE 3Effect of pH on Gel Formation of Tetra-amino PEG/Tetra SE-PEGFormulations pH of pH of pH of Tetra-amino Tetra Resulting Gelation PEGSE-PEG Mixture Gelation Time Temp. 10 4.1 6.9 10-15 minutes 45° C. 107.0 7.2 <5 minutes 45° C.

[0192] Extrudability through a 27-gauge needle was evaluated for gelscomprising tetra-amino PEG and 1 - 3% (by weight) tetra SE-PEG. The gelswere contained within 1-cc syringes. The force required to depress thesyringe plunger at a rate of 5 centimeters per minute was measured usingthe Instron Universal Tester, Model 4202. Results of extrusion testingare presented in Table 4, below. TABLE 4 Extrusion of Tetra-aminoPEG/Tetra SE-PEG Gels Through a 27-Gauge Needle Concentration of SE-PEG(by weight) Extrusion Force (N) 1.5-2% 10-11   2-2.5% 52 2.5-3% 88

[0193] Extrusion forces of 100 N or less are considered acceptable formanual injection without the aid of a syringe assist device.

[0194] Tensile strength (i.e., elasticity) of 3 mm thick gels comprisingtetra-amino PEG and 2.5, 5, and 10% (by weight) tetra SE-PEG wasmeasured using the Instron Universal Tester, Model 4202. Gels of varyinginitial lengths were stretched at a rate of 10 millimeters per minute.Length of each gel, strain at failure (change in length as a percentageof the initial length), and force at failure are set forth in Table 5,below. TABLE 5 Tensile Strength of Tetra-amino PEG/Tetra SE-PEG GelsSE-PEG Conc. Initial Strain at Force at (wt %) Length (cm) FailureFailure (N) 10 1.4 139% 0.4 10 1.9  99% 0.5 10 2.5  78% 0.5 5 1.3 111%0.2 5 1.3  99% 0.2 5 1.6  94% 0.2 2.5 1.0 237% <0.1 2.5 1.5 187% <0.12.5 1.7 129% <0.1

[0195] Gels containing 5 and 10% tetra SE-PEG approximately doubled inlength prior to breaking. Gels containing 2.5% SE-PEG approximatelytripled in length prior to breaking, but were considerably weaker (i.e.,lower force at failure) than the more highly crosslinked gels.

EXAMPLE 6 (Effect of pH on Gel Formation of Tetra-amino PEG/Tetra SE-PEGFormulations)

[0196] Gels comprising various concentrations of tetra-amino PEG andtetra SE-PEG at pH 6, 7, and 8 were prepared in petri dishes. Followingmixing of the tetra-amino PEG and tetra SE-PEG, the dishes were tiltedrepeatedly; the gelation time was considered to be the point at whichthe formulation ceased to flow. The effect of pH on gelation time of thevarious tetra-amino PEG / tetra SE-PEG formulations at room temperatureis shown in Table 6, below. TABLE 6 Effect of pH on Gel Formation ofTetra-amino PEG/Tetra SE-PEG Formulations Tetra-amino PEG Tetra SE-PEGConc. (mg/ml) Conc. (mg/ml) pH Gelation Time 20 20 6 >90.0 min 20 20 720.0 min 20 20 8 1.4 min 50 50 6 24.0 min 50 50 7 3.5 min 50 50 8 10.0sec 100 100 6 9.0 min 100 100 7 47.0 sec 100 100 8 10.0 sec 200 200 62.0 min 200 200 7 9.0 sec 200 200 8 5.0 sec

[0197] The time required for gel formation decreased with increasing pHand increasing tetra-amino PEG and tetra SE-PEG concentrations.

EXAMPLE 7 (Culturing of Cells in Crosslinked Multi-amino PEG Matrix)

[0198] Thirty (30) milligrams of tetra-amino PEG (10,000 MW, obtainedfrom Shearwater Polymers, Huntsville, Ala.) was dissolved in 0.6 ml PBS,then sterile filtered. Thirty (30) milligrams of tetrafunctionallyactivated SE-PEG (“tetra SE- PEG”, 10,000 MW, also obtained fromShearwater Polymers) was dissolved in 0.6 ml of PBS, then sterilefiltered.

[0199] The solutions of tetra-amino PEG and tetra SE-PEG were mixedtogether with a pellet containing human skin fibroblast (“HSF” ) cells(CRL #1885, passage 4, obtained from American Tissue Type CultureCollection, Rockville, Md.). Two hundred fifty (250) microliters of theresulting cell-containing tetra-amino PEG / tetra SE-PEG (PEG-PEG)solution was dispensed into each of two wells on a 48-well culture plateand allowed to gel for approximately 5 minutes at room temperature. One(1) milliter of Dulbecco Modified Eagle's Media (supplemented with 10%fetal bovine serum, L-glutamine, penicillin-streptomycin, andnon-essential amino acids) was added to each of the two wells. Theconcentration of cells was approximately 3×10⁵ cells per milliliter oftetra- amino PEG / tetra SE-PEG solution, or 7.5×10⁵ cells per well.

[0200] To prepare a control, a pellet of HSF cells were suspended in 1.2ml of complete media. Two hundred fifty (250) microliters of the controlmixture was dispensed into each of three wells on the same 48-wellculture plate as used above. Each well was estimated to containapproximately 7.5×10⁵ cells. Each well was given fresh media every otherday.

[0201] Initially, the cell-containing tetra-amino PEG tetra SE-PEG gelswere clear and the cells were found to be densely populated andspheroidal in morphology, indicating that there was little adhesionbetween the cells and the PEG/PEG gel (the cells would normally assume aflattened, spindle-shaped morphology when adhered to a substrate, suchas to the treated plastic of the tissue culture plates). After three 3days incubation at 37° C., the media in the wells containing the PEG/PEGgels was found to have lightened in color (Dulbecco Modified Eagle'sMedia is normally red in color), indicating a pH change in the media.This indicated that the cells were alive and feeding. At 7 daysincubation at 37° C., the cells were still spheroidal in morphology(indicating lack of adhesion to the gel) and the media had lightenedeven further, indicating that the cells were still viable and continuedto feed.

[0202] On day 7, the contents of each well were placed in a 10% formalinsolution for histological evaluation. According to histologicalevaluation, an estimated 75% of the cells in the wells containing thePEG/PEG gels appeared to be alive, but did not appear to be reproducing.

[0203] The results of the experiment indicate that HSF cells are viablein the tetra- amino PEG/tetra SE-PEG crosslinked gels, but did not seemto adhere to the gel and did not appear to reproduce while entrappedwithin the gel matrix. As described above, adherence or non-adherence ofcells to a substrate material can influence the cells' morphology. Incertain types of cells, cellular morphology can, in turn, influencecertain cellular functions. Therefore, non-adherence of the cells to thePEG - PEG gel matrix may be an advantage in the delivery of particularcell types whose function is influenced by cell morphology. For example,the ability of cartilage cells to produce extracellular matrix materialsis influenced by cellular morphology: when the cells are in theflattened, spindle- shaped configuration, the cells are in reproductivemode; when the cells are in the spheroidal configuration, reproductionstops, and the cells begin to produce extracellular matrix components.

[0204] Because the PEG - PEG gels are not readily degraded in vivo, thegels may be particularly useful in cell delivery applications where itis desirable that the cells remain entrapped within the matrix forextended periods-of time:

What is claimed is:
 1. A composition comprising a first syntheticpolymer having nucleophilic groups, and a second synthetic polymerhaving electrophilic groups, wherein said nucleophilic groups and saidelectrophylic groups are capable of reacting to form covalent bondsbetween the first synthetic polymer and the second synthetic polymerwhich results in formation of a three-dimensional matrix.
 2. Thecomposition of claim 1 , wherein said first synthetic polymer has mnucleophilic groups, and said second synthetic polymer has nnucleophilic groups, wherein m and n are each greater than or equal to2, and wherein m+n is greater than or equal to
 5. 3. The composition ofclaim 2 , wherein m is greater than or equal to two, and n=2.
 4. Thecomposition of claim 2 wherein m=2, and n is greater than or equal totwo.
 5. The composition of claim 2 , wherein m and n are each greaterthan or equal to three.
 6. The composition of claim 1 , wherein thefirst synthetic polymer is a synthetic polypeptide that contains two ormore nucleophilic groups selected from a primary amino group and a thiolgroup.
 7. The composition of claim 6 , wherein the first syntheticpolymer is a synthetic polypeptide that contains two or more lysineresidues.
 8. The composition of claim 7 , wherein the syntheticpolypeptide is a poly(lysine).
 9. The composition of claim 6 , whereinthe first synthetic polymer is a synthetic polypeptide that contains twoor more cysteine residues.
 10. The composition of claim 1 , wherein thefirst synthetic polymer is a polyethylene glycol that has been modifiedto contain two or more nucleophilic groups selected from a primary aminogroup and a thiol group.
 11. The composition of claim 1 , wherein thesecond synthetic polymer is a synthetic hydrophilic polymer containingtwo or more electrophilic groups.
 12. The composition of claim 10 ,wherein the synthetic hydrophilic polymer contains two or moresuccinimidyl groups.
 13. The composition of claim 10 , wherein thesynthetic hydrophilic polymer is a polyethylene glycol derivative. 14.The composition of claim 1 , wherein the second synthetic polymer is asynthetic hydrophobic polymer containing two or more succinimidylgroups.
 15. The composition of claim 11 , wherein the synthetichydrophobic polymer is selected from the group consisting of:disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate,dithiobis(succinimidylpropionate), bis(2-succinimidooxycarbonyloxy)ethyl sulfone, 3,3′-dithiobis(sulfosuccinimidylpropionate), and their analogs and derivatives. 16.The composition of claim 13 , wherein the synthetic hydrophobic polymerhas been chemically derivatized to contain two or more succinimidylgroups.
 17. The composition of claim 15 , wherein the hydrophobicpolymer is a polyacid.
 18. The composition of claim 16 , wherein thepolyacid is selected from the group consisting of:trimethylolpropane-based tricarboxylic acid, di(trimethylolpropane)-based tetracarboxylic acid, heptanedioic acid, octanedioicacid, and hexadecanedioic acid.
 19. The composition of claim 1 furthercomprising a polysaccharide or a protein.
 20. The composition of claim18 , wherein the polysaccharide is a glycosaminoglycan.
 21. Thecomposition of claim 19 , wherein the glycosaminoglycan is selected fromthe group consisting of: hyaluronic acid, chitin, chondroitin sulfate A,chondroitin sulfate B, chondroitin sulfate C, keratin sulfate,keratosulfate, heparin, and derivatives thereof.
 22. The composition ofclaim 18 , wherein the protein is collagen or a derivative thereof. 23.A composition comprising a first polyethylene glycol having primaryamino groups, and a second polyethylene glycol having succinimidylgroups.
 24. The composition of claim 23 , wherein said firstpolyethylene glycol has m primary amino groups, and said secondpolyethylene glycol has n succinimidyl groups, wherein m and n are eachgreater than or equal to 2, and wherein m+n is greater than or equal to5.
 25. The composition of claim 24 , wherein m is greater than 3, andn=2.
 26. The composition of claim 24 , wherein m=2 and n is greater thanor equal to
 3. 27. The composition of claim 24 , wherein m and n areeach greater than or equal to
 3. 28. The composition of claim 23 furthercomprising a naturally occurring polysaccharide or a naturally occurringprotein.
 29. The composition of claim 28 , wherein the naturallyoccurring polysaccharide is a glycosaminoglycan.
 30. The composition ofclaim 29 , wherein the glycosaminoglycan is selected from the followinggroup: hyaluronic acid, chitin, chondroitin sulfate A, chondroitinsulfate B, chondroitin sulfate C, keratin sulfate, keratosulfate,heparin, and derivatives thereof.
 31. The composition of claim 28 ,wherein the naturally occurring protein is collagen or a derivativethereof.
 32. A method for effecting the nonsurgical attachment of afirst surface to a second surface, comprising the steps of: providing afirst synthetic polymer containing nucleophilic groups and a secondsynthetic polymer containing electrophilic groups; mixing the firstsynthetic polymer and the second synthetic polymer to initiatecrosslinking; applying the mixture to a first surface before substantialcrosslinking has occurred; and contacting the first surface with asecond surface to effect adhesion between the first surface and thesecond surface.
 33. The method of claim 32 , wherein said firstsynthetic polymer has m nucleophilic groups and said second syntheticpolymer has n electrophilic groups, wherein m and n are each greaterthan or equal to 2, and wherein m+n is greater than or equal to
 5. 34.The method of claim 32 , wherein one of the first and second surfaces isa native tissue surface and the other of the first and second surfacesis selected from a non-native tissue surface and a surface of asynthetic implant.
 35. The method of claim 32 , wherein both the firstand second surfaces are native tissue surfaces.
 36. A method forintroducing a crosslinked synthetic polymer composition into a tissuewithin a body of a mammalian subject, comprising the steps of: providinga first synthetic polymer containing nucleophilic groups and a secondsynthetic polymer containing electrophilic groups; administering thefirst synthetic polymer and the second synthetic polymer simultaneouslyto the tissue; and allowing the first synthetic polymer and the secondsynthetic polymer to crosslink in situ.
 37. The method of claim 36 ,wherein said first synthetic polymer has m nucleophilic groups and saidsecond synthetic polymer has n electrophilic groups, wherein m and n areeach greater than or equal to 2, and wherein m+n is greater than orequal to
 5. 38. The method of claim 36 , wherein the tissue is softtissue.
 39. The method of claim 36 , wherein the tissue is hard tissue.40. The method of claim 36 , wherein the first synthetic polymer and thesecond synthetic polymer are contained within separate barrels of andadministered from a dual-compartment syringe.
 41. The method of claim 36comprising the additional step of forming a mixture by mixing the firstsynthetic polymer and the second synthetic polymer beforeadministration, wherein the mixture is administered within 60 seconds ofmixing.
 42. A method for preventing the adhesion of a first tissue and asecond tissue, comprising the steps of: providing a first syntheticpolymer containing nucleophilic groups and a second synthetic polymercontaining electrophilic groups; forming a mixture by mixing the firstsynthetic polymer and the second synthetic polymer to initiatecrosslinking; applying the mixture to the first tissue beforesubstantial crosslinking has occurred; and allowing the first syntheticpolymer and the second synthetic polymer to continue crosslinking insitu.
 43. The method of claim 42 , wherein said first synthetic polymerhas m nucleophilic groups and said second synthetic polymer has nelectrophilic groups, wherein m and n are each greater than or equal to2, and wherein m+n is greater than or equal to
 5. 44. A method forcoating a surface of a synthetic implant, comprising the steps of:providing a first synthetic polymer containing nucleophilic groups and asecond synthetic polymer containing electrophilic groups; forming amixture by mixing the first synthetic polymer and the second syntheticpolymer to initiate crosslinking; applying the mixture to a surface of asynthetic implant; and allowing the first synthetic polymer and thesecond synthetic polymer to crosslink with each other on the surface ofthe synthetic implant.
 45. The method of claim 44 , wherein said firstsynthetic polymer has m nucleophilic groups and said second syntheticpolymer has n electrophilic groups, wherein m and n are each greaterthan or equal to 2, and wherein m+n is greater than or equal to
 5. 46.The method of claim 44 , wherein the synthetic implant is selected fromthe group consisting of: artificial blood vessels, artificial heartvalves, vascular grafts, vascular stents, vascular stent/graftcombinations, surgical membranes, surgical meshes, and breast implants.47. The method of claim 44 , wherein the mixture has a net neutralcharge.
 48. A method for preparing a negatively chargedcompound-containing matrix useful for delivery of a negatively chargedcompound to a mammalian subject, comprising the steps of: providing afirst synthetic polymer containing nucleophilic groups and a secondsynthetic polymer containing electrophilic groups; forming a mixture bymixing the first synthetic polymer and the second synthetic polymer toinitiate crosslinking, wherein the first synthetic polymer is present inthe mixture in molar excess compared to the second synthetic polymer;allowing the first synthetic polymer and the second synthetic polymer tocontinue crosslinking to form a positively charged crosslinked syntheticpolymer matrix; and reacting the matrix with the negatively chargedcompound.
 49. The method of claim 48 , wherein said first syntheticpolymer has m nucleophilic groups and said second synthetic polymer hasn electrophilic groups, wherein m and n are each greater than or equalto 2, and wherein m+n is greater than or equal to
 5. 50. The method ofclaim 48 , wherein the first synthetic polymer is a polyethylene glycol,and wherein the nucleophilic groups are selected from a primary aminogroup and a thiol group.
 51. The method of claim 48 , wherein the secondsynthetic polymer is a polyethylene glycol derivative, and wherein theelectrophilic groups are succinimidyl groups.
 52. The method of claim 48, wherein the negatively charged compound is succinylated collagen. 53.The method of claim 48 , wherein the negatively charged compound is anegatively charged glycosaminoglycan derivative selected from the groupconsisting of: sodium hyaluronate, keratan sulfate, keratosulfate,sodium chondroitin sulfate A, sodium dermatan sulfate B, sodiumchondroitin sulfate C, heparin, esterified chondroitin sulfate C,esterified heparin, and combinations thereof.
 54. A method for preparinga positively charged compound-containing matrix useful for delivery of apositively charged compound to a mammalian subject, comprising the stepsof: providing a first synthetic polymer containing nucleophilic groupsand a second synthetic polymer containing electrophilic groups; forminga mixture by mixing the first synthetic polymer and the second syntheticpolymer to initiate crosslinking, wherein the second synthetic polymeris present in the mixture in molar excess compared to the secondsynthetic polymer; allowing the first synthetic polymer and the secondsynthetic polymer to continue crosslinking to form a negatively chargedcrosslinked synthetic polymer matrix; and reacting the matrix with thepositively charged compound.
 55. The method of claim 54 , wherein saidfirst synthetic polymer has m nucleophilic groups and said secondsynthetic polymer has n electrophilic groups, wherein m and n are eachgreater than or equal to 2, and wherein m+n is greater than or equal to5.
 56. The method of claim 54 , wherein the first synthetic polymer is apolyethylene glycol, and wherein the nucleophilic groups are selectedfrom a primary amino group and a thiol group.
 57. The method of claim 54, wherein the second synthetic polymer is a polyethylene glycol, andwherein the electrophilic groups are succinimidyl groups.
 58. The methodof claim 54 , wherein the positively charged compound is methylatedcollagen.
 59. The method of claim 54 , wherein the positively chargedcompound is a glycosaminoglycan derivative selected from the groupconsisting of: esterified deacetylated hyaluronic acid, esterifieddeacetylated desulfated chondroitin sulfate A, esterified deacetylateddesulfated chondroitin sulfate C, deacetylated desulfated keratansulfate, deacetylated desulfated keratosulfate, esterified desulfatedheparin, chitosan, and combinations thereof.
 60. A method for making asynthetic lenticule, comprising the steps of: providing a firstsynthetic polymer containing nucleophilic groups and a second syntheticpolymer containing electrophilic groups; forming a mixture by mixing thefirst synthetic polymer and the second synthetic polymer to initiatecrosslinking; placing said mixture into a lenticular shaped mold or ontoa surface of an eye; and allowing the first synthetic polymer and thesecond synthetic polymer to continue crosslinking to form a clearlenticule.
 61. The method of claim 60 , wherein said first syntheticpolymer has m nucleophilic groups and said second synthetic polymer hasn electrophilic groups, wherein m and n are each greater than or equalto 2, and wherein m+n is greater than or equal to
 5. 62. The method ofclaim 60 , wherein the first synthetic polymer is a polyethylene glycol,and wherein the nucleophilic groups are selected from a primary aminogroup and a thiol group.
 63. The method of claim 60 , wherein the secondsynthetic polymer is a polyethylene glycol derivative, and wherein theelectrophilic groups are succinimidyl groups.
 64. The method of claim 60further comprising a protein.
 65. The method of claim 64 wherein saidprotein is collagen or a derivative thereof.